Chlamydia antigens and corresponding DNA fragments and uses thereof

ABSTRACT

The present invention provides a method of nucleic acid, including DNA, immunization of a host, including humans, against disease caused by infection by a strain of Chlamydia, specifically  C. pneumoniae , employing a vector containing a nucleotide sequence encoding an omp P6 precursor of a strain of  Chlamydia pneumoniae  and a promoter to effect expression of the omp P6 precursor in the host. Modifications are possible within the scope of this invention.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/171,525, filed Dec. 22, 1999, the content of which isherein incorporated by reference.

FIELD OF INVENTION

[0002] The present invention relates to the Chlamydia omp P6 precursorand corresponding DNA molecules, which can be used to prevent and treatChlamydia infection in mammals, such as humans.

BACKGROUND OF THE INVENTION

[0003] Chlamydiae are prokaryotes. They exhibit morphologic andstructural similarities to gram-negative bacteria including a trilaminarouter membrane, which contains lipopolysaccharide and several membraneproteins that are structurally and functionally analogous to proteinsfound in E coli. They are obligate intra-cellular parasites with aunique biphasic life cycle consisting of a metabolically inactive butinfectious extracellular stage and a replicating but non-infectiousintracellular stage. The replicative stage of the life-cycle takes placewithin a membrane-bound inclusion which sequesters the bacteria awayfrom the cytoplasm of the infected host cell.

[0004]C. pneumoniae is a common human pathogen, originally described asthe TWAR strain of Chlamydia psittaci but subsequently recognised to bea new species. C. pneumoniae is antigenically, genetically andmorphologically distinct from other chlamydia species (C. trachomatis,C. pecorum and C. psittaci). It shows 10% or less DNA sequence homologywith either of C. trachomatis or C.psittaci.

[0005]C. pneumoniae is the third most common cause of community acquiredpneumonia, only less frequent than Streptococcus pneumoniae andMycoplasma pneumoniae (Grayston et al. (1995) Journal of InfectiousDiseases 168:1231; Campos et al. (1995) Investigation of Ophthalmologyand Visual Science 36:1477). It can also cause upper respiratory tractsymptoms and disease, including bronchitis and sinusitis (Grayston etal. (1995) Journal of Infectious Diseases 168:1231; Grayston et al(1990) Journal of Infectious Diseases 161:618-625; Marrie (1993)Clinical Infectious Diseases. 18:501-513; Wang et al (1986) Chlamydialinfections Cambridge University Press, Cambridge. p. 329. The greatmajority of the adult population (over 60%) has antibodies to C.pneumoniae (Wang et al (1986) Chlamydial infections. CambridgeUniversity Press, Cambridge. p. 329), indicating past infection whichwas unrecognized or asymptomatic.

[0006]C. pneumoniae infection usually presents as an acute respiratorydisease (i.e., cough, sore throat, hoarseness, and fever; abnormal chestsounds on auscultation). For most patients, the cough persists for 2 to6 weeks, and recovery is slow. In approximately 10% of these cases,upper respiratory tract infection is followed by bronchitis orpneumonia. Furthermore, during a C. pneumoniae epidemic, subsequentco-infection with pneumococcus has been noted in about half of thesepneumonia patients, particularly in the infirm and the elderly. As notedabove, there is more and more evidence that C. pneumoniae infection isalso linked to diseases other than respiratory infections.

[0007] The reservoir for the organism is presumably people. In contrastto C. psittaci infections, there is no known bird or animal reservoir.Transmission has not been clearly defined. It may result from directcontact with secretions, from fomites, or from airborne spread. There isa long incubation period, which may last for many months. Based onanalysis of epidemics, C. pneumoniae appears to spread slowly through apopulation (case-to-case interval averaging 30 days) because infectedpersons are inefficient transmitters of the organism. Susceptibility toC. pneumoniae is universal. Reinfections occur during adulthood,following the primary infection as a child. C. pneumoniae appears to bean endemic disease throughout the world, noteworthy for superimposedintervals of increased incidence (epidemics) that persist for 2 to 3years. C. trachomatis infection does not confer cross-immunity to C.pneumoniae. Infections are easily treated with oral antibiotics,tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). Arecently developed drug, azithromycin, is highly effective as asingle-dose therapy against chlamydial infections.

[0008] In most instances, C. pneumoniae infection is often mild andwithout complications, and up to 90% of infections are subacute orunrecognized. Among children in industrialized countries, infectionshave been thought to be rare up to the age of 5 y, although a recentstudy (E Normann et al, Chlamydia pneumoniae in children with acuterespiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp23-27) has reported that many children in this age group show PCRevidence of infection despite being seronegative, and estimates aprevalence of 17-19% in 2-4 y olds. In developing countries, theseroprevalence of C. pneumoniae antibodies among young children iselevated, and there are suspicions that C. pneumoniae may be animportant cause of acute lower respiratory tract disease and mortalityfor infants and children in tropical regions of the world.

[0009] From seroprevalence studies and studies of local epidemics, theinitial C. pneumoniae infection usually happens between the ages of 5and 20 y. In the USA, for example, there are estimated to be 30,000cases of childhood pneumonia each year caused by C. pneumoniae.Infections may cluster among groups of children or young adults (e.g.,school pupils or military conscripts).

[0010]C. pneumoniae causes 10 to 25% of community-acquired lowerrespiratory tract infections (as reported from Sweden, Italy, Finland,and the USA). During an epidemic, C. pneumonia infection may account for50 to 60% of the cases of pneumonia. During these periods, also, moreepisodes of mixed infections with S. pneumoniae have been reported.

[0011] Reinfection during adulthood is common; the clinical presentationtends to be milder. Based on population seroprevalence studies, theretends to be increased exposure with age, which is particularly evidentamong men. Some investigators have speculated that a persistent,asymptomatic C. pneumoniae infection state is common.

[0012] In adults of middle age or older, C. pneumoniae infection mayprogress to chronic bronchitis and sinusitis. A study in the USArevealed that the incidence of pneumonia caused by C. pneumoniae inpersons younger than 60 years is 1 case per 1,000 persons per year; butin the elderly, the disease incidence rose three-fold. C. pneumoniaeinfection rarely leads to hospitalization, except in patients with anunderlying illness. of considerable importance is the association ofatherosclerosis and C. pneumoniae infection. There are severalepidemiological studies showing a correlation of previous infectionswith C. pneumoniae and heart attacks, coronary artery and carotid arterydisease (Saikku et al. (1988) Lancet;ii:983-986; Thom et al. (1992) JAMA268:68-72; Linnanmaki et al. (1993), Circulation 87:1030; Saikku et al.(1992) Annals Internal Medicine 116:273-287; Melnick et al(1993)American Journal of Medicine 95:499). Moreover, the organisms has beendetected in atheromas and fatty streaks of the coronary, carotid,peripheral arteries and aorta (Shor et al. (1992) South African. MedicalJournal 82:158-161; Kuo et al. (1993) Journal of Infectious Diseases167:841-849; Kuo et al. (1993) Arteriosclerosis and Thrombosis13:1501-1504; Campbell et al (1995) Journal of Infectious Diseases172:585; Chiu et al. Circulation, 1997. Circulation. 96:2144-2148).Viable C. pneumoniae has been recovered from the coronary and carotidartery (Ramirez et al (1996) Annals of Internal Medicine 125:979-982;Jackson et al. 1997. J. Infect. Dis. 176:292-295). Furthermore, it hasbeen shown that C. pneumoniae can induce changes of atherosclerosis in arabbit model (Fong et al. 1997. Journal of Clinical Microbiolology 35:48and Laitinen et al. 1997. Infect. Immun. 65:4832-4835). Taken together,these results indicate that it is highly probable that C. pneumoniae cancause atherosclerosis in humans, though the epidemiological importanceof chlamydial atherosclerosis remains to be demonstrated.

[0013] A number of recent studies have also indicated an associationbetween C. pneumoniae infection and asthma. Infection has been linked towheezing, asthmatic bronchitis, adult-onset asthma and acuteexacerbations of asthma in adults, and small-scale studies have shownthat prolonged antibiotic treatment was effective at greatly reducingthe severity of the disease in some individuals (Hahn DL, et al.Evidence for Chlamydia pneumoniae infection in steroid-dependent asthma.Ann Allergy Asthma Immunol. 1998 Jan; 80(1): 45-49.; Hahn DL, et al.Association of Chlamydia pneumoniae IgA antibodies with recentlysymptomatic asthma. Epidemiol Infect. 1996 December; 117(3): 513-517;Bjornsson E, et al. Serology of chlamydia in relation to asthma andbronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.;Hahn DL. Treatment of Chlamydia pneumoniae infection in adult asthma: abefore-after trial. J Fam Pract. 1995 Oct; 41(4): 345-351.; Allegra L,et al. Acute exacerbations of asthma in adults: role of Chlamydiapneumoniae infection. Eur Respir J. 1994 Dec; 7(12): 2165-2168.; Hahn DL, et al. Association of Chlamydia pneumoniae (strain TWAR) infectionwith wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA. 1991Jul 10; 266(2): 225-230).

[0014] In light of these results a protective vaccine against C.pneumoniae infection would be of considerable importance. There is notyet an effective vaccine for any human chlamydial infection. It isconceivable that an effective vaccine can be developed using physicallyor chemically inactivated Chlamydiae. However, such a vaccine does nothave a high margin of safety. In general, safer vaccines are made bygenetically manipulating the organism by attenuation or by recombinantmeans. Accordingly, a major obstacle in creating an effective and safevaccine against human chlamydial infection has been the paucity ofgenetic information regarding Chlamydia, specifically C. pneumoniae.

[0015] Studies with C. trachomatis and C. psittaci indicate that safeand effective vaccine against Chlamydia is an attainable goal. Forexample, mice which have recovered from a lung infection with C.trachomatis are protected from infertility induced by a subsequentvaginal challenge (Pal et al.(1996) Infection and Immunity.64:5341).Similarly, sheep immunized with inactivated C. psittaci were protectedfrom subsequent chlamydial-induced abortions and stillbirths (Jones etal. (1995) Vaccine 13:715). In a mouse model, protection from chlamydialinfections has been associated with Thl immune responses, particularlyCD8+ CTL response (Rottenberg et al. 1999. J. Immunol. 162:2829-2836 andPenttila et al. 1999. Immunology. 97:490-496) and it is unlikely thatsimilar responses will need to be induced in humans to conferprotection. However, antigens able to elicit a protective immuneresponse against C. pneumoniae are largely unknown. The presence ofsufficiently high titres of neutralising antibody at mucosal surfacescan also exert a protective effect (Cotter et al. (1995) Infection andImmunity 63:4704).

[0016] Antigenic variation within the species C. pneumoniae is not welldocumented due to insufficient genetic information, though variation isexpected to exist based on C. trachomatis. Serovars of C. trachomatisare defined on the basis of antigenic variation in the major outermembrane protein (MOMP), but published C. pneumoniae MOMP gene sequencesshow no variation between several diverse isolates of the organism(Campbell et al. Infection and Immunity (1990) 58:93; McCafferty et alInfection and Immunity (1995) 63:2387-9; Gaydos et al. Infection andImmunity.(1992) 60(12):5319-5323). The gene encoding a 76 kDa antigenhas been cloned from a single strain of C. pneumoniae and the sequencepublished (Perez Melgosa et al. Infection and Immunity.(1994) 62:880).An operon encoding the 9 kDa and 60 kDa cyteine-rich outer membraneprotein genes has been described (Watson et al., Nucleic Acids Res(1990) 18:5299; Watson et al., Microbiology (1995) 141:2489). Manyantigens recognized by immune sera to C. pneumoniae are conserved acrossall chlamydiae, but 98 kDa, 76 kDa and several other proteins may be C.pneumoniae-specific (Knudsen et al. Infect. Immun. 1999. 67:375-383;Perez Melgosa et al. Infection and Immunity. 1994. 62:880; Melgosa etal., FEMS Microbiol Lett 1993. 112 :199;, Campbell et al., J. Clin.Microbiol. 1990. 28 :1261; Iijima et al., J. Clin. Microbiol. 1994.32:583). Antisera to 76 kDa and 54 kDa antigens have been reported toneutralize C. pneumoniae in vitro (Perez Melgosa et al. 1994. Infect.Immun. 62:880-886 and Wiedman-Al-Ahmad et al. 1997. Clin. Diagn. Lab.Immunol. 4:700-704). An assessment of the number and relative frequencyof any C. pneumoniae serotypes, and the defining antigens, is not yetpossible. The entire genome sequence of C. pneumoniae strain CWL-029 isnow known (http://chlamydia-www.berkeley.edu:4231/) and as furthersequences become available a better understanding of antigenic variationmay be gained.

[0017] Many antigens recognised by immune sera to C. pneumoniae areconserved across all chlamydiae, but 98kDa, 76 kDa and 54 kDa proteinsappear to be C. pneumoniae-specific (Campos et al. (1995) Investigationof Ophthalmology and Visual Science 36:1477; Marrie (1993) ClinicalInfectious Diseases. 18:501-513; Wiedmann-Al-Ahmad M, et al. Reactionsof polyclonal and neutralizing anti-p54 monoclonal antibodies with anisolated, species-specific 54-kilodalton protein of Chlamydiapneumoniae. Clin Diagn Lab Immunol. 1997 November; 4(6): 700-704).

[0018] Immunoblotting of isolates with sera from patients does showvariation of blotting patterns between isolates, indicating thatserotypes C. pneumoniae may exist (Grayston et al. (1995) Journal ofInfectious Diseases 168:1231; Ramirez et al (1996) Annals of InternalMedicine 125:979-982). However, the results are potentially confoundedby the infection status of the patients, since immunoblot profiles of apatient's sera change with time post-infection. An assessment of thenumber and relative frequency of any serotypes, and the definingantigens, is not yet possible.

[0019] The use of DNA immunization to elicit a protective immuneresponse in Balb/c mice against pulmonary infection with the mousepneumonitis (MoPn) strain of Chlamydia trachomatis has recently beendescribed (Zhang et al. 1997. J. Infect. Dis. 76:1035-1040 and Zhang etal. 1999. Immunology. 96:314-321).

[0020] Accordingly, a need exists for identifying and isolatingpolynucleotide sequences of C. pneumoniae for use in preventing andtreating Chlamydia infection.

SUMMARY OF THE INVENTION

[0021] The present invention provides purified and isolatedpolynucleotide molecules that encode the Chlamydia polypeptidesdesignated omp P6 precursor gene product (SEQ ID No: 1) which can beused in methods to prevent, treat, and diagnose Chlamydia infection. Inone form of the invention, the polynucleotide molecules are DNA thatencode the polypeptide of SEQ ID No: 2.

[0022] Another form of the invention provides polypeptides correspondingto the isolated DNA molecules. The amino acid sequence of thecorresponding encoded polypeptide is shown as SEQ ID No: 2.

[0023] Those skilled in the art will readily understand that theinvention, having provided the polynucleotide sequences encoding theChlamydia omp P6 precursor, also provides polynucleotides encodingfragments derived from such a polypeptide. Moreover, the invention isunderstood to provide mutants and derivatives of such polypeptides andfragments derived therefrom, which result from the addition, deletion,or substitution of non-essential amino acids as described herein. Thoseskilled in the art would also readily understand that the invention,having provided the polynucleotide sequences encoding Chlamydiapolypeptides, further provides monospecific antibodies that specificallybind to such polypeptides.

[0024] The present invention has wide application and includesexpression cassettes, vectors, and cells transformed or transfected withthe polynucleotides of the invention. Accordingly, the present inventionfurther provides (i) a method for producing a polypeptide of theinvention in a recombinant host system and related expression cassettes,vectors, and transformed or transfected cells; (ii) a vaccine, or a livevaccine vector such as a pox virus, Salmonella typhimurium, or Vibriocholerae vector, containing a polynucleotide of the invention, suchvaccines and vaccine vectors being useful for, e.g., preventing andtreating Chlamydia infection, in combination with a diluent or carrier,and related pharmaceutical compositions and associated therapeuticand/or prophylactic methods; (iii) a therapeutic and/or prophylactic useof an RNA or DNA molecule of the invention, either in a naked form orformulated with a delivery vehicle, a polypeptide or combination ofpolypeptides, or a monospecific antibody of the invention, and relatedpharmaceutical compositions; (iv) a method for diagnosing the presenceof Chlamydia in a biological sample, which can involve the use of a DNAor RNA molecule, a monospecific antibody, or a polypeptide of theinvention; and (v) a method for purifying a polypeptide of the inventionby antibody-based affinity chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further understood from thefollowing description with reference to the drawings, in which:

[0026]FIG. 1 shows the nucleotide sequence of the omp P6 precursor gene(SEQ ID No: 1) and the deduced amino acid sequence of the omp P6precursor gene product from Chlamydia pneumoniae (SEQ ID No: 2).

[0027]FIG. 2 shows the restriction enzyme analysis of the C. pneumoniaeomp P6 precursor gene.

[0028]FIG. 3 shows the construction and elements of plasmid pCABk831.

[0029]FIG. 4 illustrates protection against C. pneumoniae infection bypCABk831 following DNA immunization.

DETAILED DESCRIPTION OF INVENTION

[0030] An open reading frame (ORF) encoding the Chlamydial omp P6precursor has been identified from the C. pneumoniae genome. The geneencoding this protein has been inserted into an expression plasmid andshown to confer immune protection against chlamydial infection.Accordingly, this omp P6 precursor and related polypeptides can be usedto prevent and treat Chlamydia infection.

[0031] According to a first aspect of the invention, isolatedpolynucleotides are provided which encode Chlamydia polypeptides, whoseamino acid sequences are shown in SEQ ID No: 2.

[0032] The term “isolated polynucleotide” is defined as a polynucleotideremoved from the environment in which it naturally occurs. For example,a naturally-occurring DNA molecule present in the genome of a livingbacteria or as part of a gene bank is not isolated, but the samemolecule separated from the remaining part of the bacterial genome, as aresult of, e.g., a cloning event (amplification), is isolated.Typically, an isolated DNA molecule is free from DNA regions (e.g.,coding regions) with which it is immediately contiguous at the 5′ or 3′end, in the naturally occurring genome. Such isolated polynucleotidesmay be part of a vector or a composition and still be defined asisolated in that such a vector or composition is not part of the naturalenvironment of such polynucleotide.

[0033] The polynucleotide of the invention is either RNA or DNA (cDNA,genomic DNA, or synthetic DNA), or modifications, variants, homologs orfragments thereof. The DNA is either double-stranded or single-stranded,and, if single-stranded, is either the coding strand or the non-coding(anti-sense) strand. Any one of the sequences that encode thepolypeptides of the invention as shown in SEQ ID No: 1 is (a) a codingsequence, (b) a ribonucleotide sequence derived from transcription of(a), or (c) a coding sequence which uses the redundancy or degeneracy ofthe genetic code to encode the same polypeptides. By “polypeptide” or“protein” is meant any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). Both terms are used interchangeably in the presentapplication.

[0034] Consistent with the first aspect of the invention, amino acidsequences are provided which are homologous to SEQ ID No: 2. As usedherein, “homologous amino acid sequence” is any polypeptide which isencoded, in whole or in part, by a nucleic acid sequence whichhybridizes at 25-35° C. below critical melting temperature (Tm), to anyportion of the nucleic acid sequence of SEQ ID No: 1. A homologous aminoacid sequence is one that differs from an amino acid sequence shown inSEQ ID No: 2 by one or more conservative amino acid substitutions. Sucha sequence also encompass serotypic variants (defined below) as well assequences containing deletions or insertions which retain inherentcharacteristics of the polypeptide such as immunogenicity. Preferably,such a sequence is at least 75%, more preferably 80%, and mostpreferably 90% identical to SEQ ID No: 2.

[0035] Homologous amino acid sequences include sequences that areidentical or substantially identical to SEQ ID No: 2. By “amino acidsequence substantially identical” is meant a sequence that is at least90%, preferably 95%, more preferably 97%, and most preferably 99%identical to an amino acid sequence of reference and that preferablydiffers from the sequence of reference by a majority of conservativeamino acid substitutions.

[0036] Conservative amino acid substitutions are substitutions amongamino acids of the same class. These classes include, for example, aminoacids having uncharged polar side chains, such as asparagine, glutamine,serine, threonine, and tyrosine; amino acids having basic side chains,such as lysine, arginine, and histidine; amino acids having acidic sidechains, such as aspartic acid and glutamic acid; and amino acids havingnonpolar side chains, such as glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, andcysteine.

[0037] Homology is measured using sequence analysis software such asSequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705. Amino acid sequences are aligned to maximizeidentity. Gaps may be artificially introduced into the sequence toattain proper alignment. Once the optimal alignment has been set up, thedegree of homology is established by recording all of the positions inwhich the amino acids of both sequences are identical, relative to thetotal number of positions.

[0038] Homologous polynucleotide sequences are defined in a similar way.Preferably, a homologous sequence is one that is at least 45%, morepreferably 60%, even more preferably 75%, even more preferably 85% andmost preferably 90% identical to the coding sequence of SEQ ID No: 1.

[0039] Consistent with the first aspect of the invention, polypeptideshaving a sequence homologous to SEQ ID No: 2 include naturally-occurringallelic variants, as well as mutants or any other non-naturallyoccurring variants that retain the inherent characteristics of thepolypeptide of SEQ ID No: 2.

[0040] As is known in the art, an allelic variant is an alternate formof a polypeptide that is characterized as having a substitution,deletion, or addition of one or more amino acids that does not alter thebiological function of the polypeptide. By “biological function” ismeant the function of the polypeptide in the cells in which it naturallyoccurs, even if the function is not necessary for the growth or survivalof the cells. For example, the biological function of a porin is toallow the entry into cells of compounds present in the extracellularmedium. Biological function is distinct from antigenic property. Apolypeptide can have more than one biological function.

[0041] Allelic variants are very common in nature. For example, abacterial species such as C. pneumoniae, is usually represented by avariety of strains that differ from each other by minor allelicvariations. Indeed, a polypeptide that fulfills the same biologicalfunction in different strains can have an amino acid sequence (andpolynucleotide sequence) that is not identical in each of the strains.Despite this variation, an immune response directed generally againstmany allelic variants has been demonstrated. In studies of theChlamydial MOMP antigen, cross-strain antibody binding plusneutralization of infectivity occurs despite amino acid sequencevariation of MOMP from strain to strain, indicating that the MOMP, whenused as an immunogen, is tolerant of amino acid variations.

[0042] Polynucleotides encoding homologous polypeptides or allelicvariants are retrieved by polymerase chain reaction (PCR) amplificationof genomic bacterial DNA extracted by conventional methods. Thisinvolves the use of synthetic oligonucleotide primers matching upstreamand downstream of the 5′ and 3′ ends of the encoding domain. Suitableprimers are designed according to the nucleotide sequence informationprovided in SEQ ID No:1. The procedure is as follows: a primer isselected which consists of 10 to 40, preferably 15 to 25 nucleotides. Itis advantageous to select primers containing C and G nucleotides in aproportion sufficient to ensure efficient hybridization; i.e., an amountof C and G nucleotides of at least 40%, preferably 50% of the totalnucleotide content. A standard PCR reaction contains typically 0.5 to 5Units of Taq DNA polymerase per 100 μL, 20 to 200 μM deoxynucleotideeach, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesiumover the total deoxynucleotide concentration, 10⁵ to 10⁶ targetmolecules, and about 20 pmol of each primer. About 25 to 50 PCR cyclesare performed, with an annealing temperature 15° C. to 5° C. below thetrue Tm of the primers. A more stringent annealing temperature improvesdiscrimination against incorrectly annealed primers and reducesincorportion of incorrect nucleotides at the 3′ end of primers. Adenaturation temperature of 95° C. to 97° C. is typical, although highertemperatures may be appropriate for dematuration of G+C-rich targets.The number of cycles performed depends on the starting concentration oftarget molecules, though typically more than 40 cycles is notrecommended as non-specific background products tend to accumulate.

[0043] An alternative method for retrieving polynucleotides encodinghomologous polypeptides or allelic variants is by hybridizationscreening of a DNA or RNA library. Hybridization procedures arewell-known in the art and are described in Ausubel et al., (Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons Inc.,1994), Silhavy et al. (Silhavy et al. Experiments with Gene Fusions,Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis etal. A Manual for Genetic Engineering: Advanced Bacterial Genetics, ColdSpring Harbor Laboratory Press, 1980)). Important parameters foroptimizing hybridization conditions are reflected in a formula used toobtain the critical melting temperature above which two complementaryDNA strands separate from each other (Casey & Davidson, Nucl. Acid Res.(1977) 4:1539). For polynucleotides of about 600 nucleotides or larger,this formula is as follows: Tm81.5+0.41×(% G+C)+16.6 log (cation ionconcentration)−0.63×(% formamide)−600/base number. Under appropriatestringency conditions, hybridization temperature (Th) is approximately20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C. below thecalculated Tm. Those skilled in the art will understand that optimaltemperature and salt conditions can be readily determined.

[0044] For the polynucleotides of the invention, stringent conditionsare achieved for both pre-hybridizing and hybridizing incubations (i)within 4-16 hours at 42° C., in 6× SSC containing 50% formamide, or (ii)within 4-16 hours at 65° C. in an aqueous 6× SSC solution (1 M NaCl, 0.1M sodium citrate (pH 7.0)). Typically, hybridization experiments areperformed at a temperature from 60 to 68° C., e.g. 65° C. At such atemperature, stringent hybridization conditions can be achieved in 6×SSC, preferably in 2× SSC or 1× SSC, more preferably in 0.5× SSc, 0.3×SSC or 0.1× SSC (in the absence of formamide). 1× SSC contains 0.15 MNaCl and 0.015 M sodium citrate.

[0045] Useful homologs and fragments thereof that do not occur naturallyare designed using known methods for identifying regions of an antigenthat are likely to tolerate amino acid sequence changes and/ordeletions. As an example, homologous polypeptides from different speciesare compared; conserved sequences are identified. The more divergentsequences are the most likely to tolerate sequence changes. Homologyamong sequences may be analyzed using, as an example, the BLAST homologysearching algorithm of Altschul et al., Nucleic Acids Res.; 25:3389-3402(1997). Alternatively, sequences are modified such that they become morereactive to T- and/or B-cells, based on computer-assisted analysis ofprobable T- or B-cell epitopes Yet another alternative is to mutate aparticular amino acid residue or sequence within the polypeptide invitro, then screen the mutant polypeptides for their ability to preventor treat Chlamydia infection according to the method outlined below.

[0046] A person skilled in the art will readily understand that byfollowing the screening process of this invention, it will be determinedwithout undue experimentation whether a particular homolog of SEQ ID No.2 may be useful in the prevention or treatment of Chlamydia infection.The screening procedure comprises the steps:

[0047] (i) immunizing an animal, preferably mouse, with the test homologor fragment;

[0048] (ii) inoculating the immunized animal with Chlamydia; and

[0049] (iii) selecting those homologs or fragments which conferprotection against Chlamydia.

[0050] By “conferring protection” is meant that there is a reduction inseverity of any of the effects of Chlamydia infection, in comparisonwith a control animal which was not immunized with the test homolog orfragment.

[0051] Consistent with the first aspect of the invention, polypeptidederivatives are provided that are partial sequences of SEQ ID No. 2,partial sequences of polypeptide sequences homologous to SEQ ID No. 2,polypeptides derived from full-length polypeptides by internal deletion,and fusion proteins.

[0052] It is an accepted practice in the field of immunology to usefragments and variants of protein immunogens as vaccines, as all that isrequired to induce an immune response to a protein is a small (e.g., 8to 10 amino acid) immunogenic region of the protein. Various shortsynthetic peptides corresponding to surface-exposed antigens ofpathogens other than Chlamydia have been shown to be effective vaccineantigens against their respective pathogens, e.g. an 11 residue peptideof murine mammary tumor virus (Casey & Davidson, Nucl. Acid Res. (1977)4:1539), a 16-residue peptide of Semliki Forest virus (Snijders et al.,1991. J. Gen. Virol. 72:557-565), and two overlapping peptides of 15residues each from canine parvovirus (Langeveld et al., Vaccine12(15):1473-1480, 1994).

[0053] Accordingly, it will be readily apparent to one skilled in theart, having read the present description, that partial sequences of SEQID No: 2 or their homologous amino acid sequences are inherent to thefull-length sequences and are taught by the present invention. Suchpolypeptide fragments preferably are at least 12 amino acids in length.Advantageously, they are at least 20 amino acids, preferably at least 50amino acids, more preferably at least 75 amino acids, and mostpreferably at least 100 amino acids in length.

[0054] Polynucleotides of 30 to 600 nucleotides encoding partialsequences of sequences homologous to SEQ ID No: 2 are retrieved by PCRamplification using the parameters outlined above and using primersmatching the sequences upstream and downstream of the 5′ and 3′ ends ofthe fragment to be amplified. The template polynucleotide for suchamplification is either the full length polynucleotide homologous to SEQID No: 1, or a polynucleotide contained in a mixture of polynucleotidessuch as a DNA or RNA library. As an alternative method for retrievingthe partial sequences, screening hybridization is carried out underconditions described above and using the formula for calculating Tm.Where fragments of 30 to 600 nucleotides are to be retrieved, thecalculated Tm is corrected by subtracting (600/polynucleotide size inbase pairs) and the stringency conditions are defined by a hybridizationtemperature that is 5 to 10° C. below Tm. Where oligonucleotides shorterthan 20-30 bases are to be obtained, the formula for calculating the Tmis as follows: Tm=4×(G+C)+2 (A+T). For example, an 18 nucleotidefragment of 50% G+C would have an approximate Tm of 54° C. Shortpeptides that are fragments of SEQ ID No: 2 or its homologous sequences,are obtained directly by chemical synthesis (E. Gross and H. J.Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; ModernTechniques of Peptide Synthesis, John Wiley & Sons (1981), and M.Bodanzki, Principles of Peptide Synthesis, Springer-Verlag (1984)).

[0055] Useful polypeptide derivatives, e.g., polypeptide fragments, aredesigned using computer-assisted analysis of amino acid sequences. Thiswould identify probable surface-exposed, antigenic regions (Hughes etal., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acidsequences contained in SEQ ID No: 2, based on the product of flexibilityand hydrophobicity propensities using the program SEQSEE (Wishart DS, etal. “SEQSEE: a comprehensive program suite for protein sequenceanalysis.” Comput Appl Biosci. 1994 Apr;10(2):121-32), can revealpotential B- and T-cell epitopes which may be used as a basis forselecting useful immunogenic fragments and variants. This analysis usesa reasonable combination of external surface features that is likely tobe recognized by antibodies. Probable T-cell epitopes for HLA-A0201 MHCsubclass may be revealed by an algorithms that emulate an approachdeveloped at the NIH (Parker KC, et al. “Peptide binding to MHC class Imolecules: implications for antigenic peptide prediction.” Immunol Res1995;14(1):34-57).

[0056] Epitopes which induce a protective T cell-dependent immuneresponse are present throughout the length of the polypeptide. However,some epitopes may be masked by secondary and tertiary structures of thepolypeptide. To reveal such masked epitopes large internal deletions arecreated which remove much of the original protein structure and exposesthe masked epitopes. Such internal deletions sometimes effect theadditional advantage of removing immunodominant regions of highvariability among strains.

[0057] Polynucleotides encoding polypeptide fragments and polypeptideshaving large internal deletions are constructed using standard methods(Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons Inc., 1994). Such methods include standard PCR, inverse PCR,restriction enzyme treatment of cloned DNA molecules, or the method ofKunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448).Components for these methods and instructions for their use are readilyavailable from various commercial sources such as Stratagene. Once thedeletion mutants have been constructed, they are tested for theirability to prevent or treat Chlamydia infection as described above.

[0058] As used herein, a fusion polypeptide is one that contains apolypeptide or a polypeptide derivative of the invention fused at the N-or C-terminal end to any other polypeptide (hereinafter referred to as apeptide tail). A simple way to obtain such a fusion polypeptide is bytranslation of an in-frame fusion of the polynucleotide sequences, i.e.,a hybrid gene. The hybrid gene encoding the fusion polypeptide isinserted into an expression vector which is used to transform ortransfect a host cell. Alternatively, the polynucleotide sequenceencoding the polypeptide or polypeptide derivative is inserted into anexpression vector in which the polynucleotide encoding the peptide tailis already present. Such vectors and instructions for their use arecommercially available, e.g. the pMal-c2 or pMal-p2 system from NewEngland Biolabs, in which the peptide tail is a maltose binding protein,the glutathione-S-transferase system of Pharmacia, or the His-Tag systemavailable from Novagen. These and other expression systems provideconvenient means for further purification of polypeptides andderivatives of the invention.

[0059] An advantageous example of a fusion polypeptide is one where thepolypeptide or homolog or fragment of the invention is fused to apolypeptide having adjuvant activity, such as subunit B of eithercholera toxin or E. coli heat-labile toxin. Another advantageous fusionis one where the polypeptide, homolog or fragment is fused to a strongT-cell epitope or B-cell epitope. Such an epitope may be one known inthe art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al.,“Antibody production to the nucleocapsid and envelope of the Hepatitis Bvirus primed by a single synthetic T cell site”, Nature. 1987.329:547-549), or one which has been identified in another polypeptide ofthe invention based on computer-assisted analysis of probable T- orB-cell epitopes. Consistent with this aspect of the invention is afusion polypeptide comprising T- or B-cell epitopes from SEQ ID No: 2 orits homolog or fragment, wherein the epitopes are derived from multiplevariants of said polypeptide or homolog or fragment, each variantdiffering from another in the location and sequence of its epitopewithin the polypeptide. Such a fusion is effective in the prevention andtreatment of Chlamydia infection since it optimizes the T- and B-cellresponse to the overall polypeptide, homolog or fragment.

[0060] To effect fusion, the polypeptide of the invention is fused tothe N-, or preferably, to the C-terminal end of the polypeptide havingadjuvant activity or T- or B-cell epitope. Alternatively, a polypeptidefragment of the invention is inserted internally within the amino acidsequence of the polypeptide having adjuvant activity. The T- or B-cellepitope may also be inserted internally within the amino acid sequenceof the polypeptide of the invention.

[0061] Consistent with the first aspect, the polynucleotides of theinvention also encode hybrid precursor polypeptides containingheterologous signal peptides, which mature into polypeptides of theinvention. By “heterologous signal peptide” is meant a signal peptidethat is not found in naturally-occurring precursors of polypeptides ofthe invention.

[0062] Polynucleotide molecules according to the invention, includingRNA, DNA, or modifications or combinations thereof, have variousapplications. A DNA molecule is used, for example, (i) in a process forproducing the encoded polypeptide in a recombinant host system, (ii) inthe construction of vaccine vectors such as poxviruses, which arefurther used in methods and compositions for preventing and/or treatingChlamydia infection, (iii) as a vaccine agent (as well as an RNAmolecule), in a naked form or formulated with a delivery vehicle and,(iv) in the construction of attenuated Chlamydia strains that canover-express a polynucleotide of the invention or express it in anon-toxic, mutated form.

[0063] Accordingly, a second aspect of the invention encompasses (i) anexpression cassette containing a DNA molecule of the invention placedunder the control of the elements required for expression, in particularunder the control of an appropriate promoter; (ii) an expression vectorcontaining an expression cassette of the invention; (iii) a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, as well as (iv) a process forproducing a polypeptide or polypeptide derivative encoded by apolynucleotide of the invention, which involves culturing a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, under conditions that allowexpression of the DNA molecule of the invention and, recovering theencoded polypeptide or polypeptide derivative from the cell culture.

[0064] A recombinant expression system is selected from procaryotic andeucaryotic hosts. Eucaryotic hosts include yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g.,COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodopterafrugiperda (SF9) cells), and plant cells. A preferred expression systemis a procaryotic host such as E. coli. Bacterial and eucaryotic cellsare available from a number of different sources including commercialsources to those skilled in the art, e.g., the American Type CultureCollection (ATCC; Rockville, Md.). Commercial sources of cells used forrecombinant protein expression also provide instructions for usage ofthe cells.

[0065] The choice of the expression system depends on the featuresdesired for the expressed polypeptide. For example, it may be useful toproduce a polypeptide of the invention in a particular lipidated form orany other form.

[0066] One skilled in the art would redily understand that not allvectors and expression control sequences and hosts would be expected toexpress equally well the polynucleotides of this invention. With theguidelines described below, however, a selection of vectors, expressioncontrol sequences and hosts may be made without undue experimentationand without departing from the scope of this invention.

[0067] In selecting a vector, the host must be chosen that is compatiblewith the vector which is to exist and possibly replicate in it.Considerations are made with respect to the vector copy number, theability to control the copy number, expression of other proteins such asantibiotic resistance. In selecting an expression control sequence, anumber of variables are considered. Among the important variable are therelative strength of the sequence (e.g. the ability to drive expressionunder various conditions), the ability to control the sequence'sfunction, compatibility between the polynucleotide to be expressed andthe control sequence (e.g. secondary structures are considered to avoidhairpin structures which prevent efficient transcription). In selectingthe host, unicellular hosts are selected which are compatible with theselected vector, tolerant of any possible toxic effects of the expressedproduct, able to secrete the expressed product efficiently if such isdesired, to be able to express the product in the desired conformation,to be easily scaled up, and to which ease of purification of the finalproduct.

[0068] The choice of the expression cassette depends on the host systemselected as well as the features desired for the expressed polypeptide.Typically, an expression cassette includes a promoter that is functionalin the selected host system and can be constitutive or inducible; aribosome binding site; a start codon (ATG) if necessary; a regionencoding a signal peptide, e.g., a lipidation signal peptide; a DNAmolecule of the invention; a stop codon; and optionally a 3′ terminalregion (translation and/or transcription terminator). The signal peptideencoding region is adjacent to the polynucleotide of the invention andplaced in proper reading frame. The signal peptide-encoding region ishomologous or heterologous to the DNA molecule encoding the maturepolypeptide and is compatible with the secretion apparatus of the hostused for expression. The open reading frame constituted by the DNAmolecule of the invention, solely or together with the signal peptide,is placed under the control of the promoter so that transcription andtranslation occur in the host system. Promoters and signal peptideencoding regions are widely known and available to those skilled in theart and include, for example, the promoter of Salmonella typhimurium(and derivatives) that is inducible by arabinose (promoter araB) and isfunctional in Gram-negative bacteria such as E. coli (as described inU.S. Pat. No. 5,028,530 and in Cagnon et al., (Cagnon et al., ProteinEngineering (1991) 4(7):843)); the promoter of the gene of bacteriophageT7 encoding RNA polymerase, that is functional in a number of E. colistrains expressing T7 polymerase (described in U.S. Pat. No. 4,952,496);OspA lipidation signal peptide ; and RlpB lipidation signal peptide(Takase et al., J. Bact. (1987) 169:5692).

[0069] The expression cassette is typically part of an expressionvector, which is selected for its ability to replicate in the chosenexpression system. Expression vectors (e.g., plasmids or viral vectors)can be chosen, for example, from those described in Pouwels et al.(Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitableexpression vectors can be purchased from various commercial sources.

[0070] Methods for transforming/transfecting host cells with expressionvectors are well-known in the art and depend on the host system selectedas described in Ausubel et al., (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc., 1994).

[0071] Upon expression, a recombinant polypeptide of the invention (or apolypeptide derivative) is produced and remains in the intracellularcompartment, is secreted/excreted in the extracellular medium or in theperiplasmic space, or is embedded in the cellular membrane. Thepolypeptide is recovered in a substantially purified form from the cellextract or from the supernatant after centrifugation of the recombinantcell culture. Typically, the recombinant polypeptide is purified byantibody-based affinity purification or by other well-known methods thatcan be readily adapted by a person skilled in the art, such as fusion ofthe polynucleotide encoding the polypeptide or its derivative to a smallaffinity binding domain. Antibodies useful for purifying byimmunoaffinity the polypeptides of the invention are obtained asdescribed below.

[0072] A polynucleotide of the invention can also be useful as avaccine. There are two major routes, either using a viral or bacterialhost as gene delivery vehicle (live vaccine vector) or administering thegene in a free form, e.g., inserted into a plasmid. Therapeutic orprophylactic efficacy of a polynucleotide of the invention is evaluatedas described below.

[0073] Accordingly, a third aspect of the invention provides (i) avaccine vector such as a poxvirus, containing a DNA molecule of theinvention, placed under the control of elements required for expression;(ii) a composition of matter comprising a vaccine vector of theinvention, together with a diluent or carrier; specifically (iii) apharmaceutical composition containing a therapeutically orprophylactically effective amount of a vaccine vector of the invention;(iv) a method for inducing an immune response against Chlamydia in amammal (e.g., a human; alternatively, the method can be used inveterinary applications for treating or preventing Chlamydia infectionof animals, e.g., cats or birds), which involves administering to themammal an immunogenically effective amount of a vaccine vector of theinvention to elicit a protective or therapeutic immune response toChlamydia ; and particularly, (v) a method for preventing and/ortreating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumonia,C. pecorum) infection, which involves administering a prophylactic ortherapeutic amount of a vaccine vector of the invention to an infectedindividual. Additionally, the third aspect of the invention encompassesthe use of a vaccine vector of the invention in the preparation of amedicament for preventing and/or treating Chlamydia infection.

[0074] As used herein, a vaccine vector expresses one or severalpolypeptides or derivatives of the invention. The vaccine vector mayexpress additionally a cytokine, such as interleukin-2 (IL-2) orinterleukin-12 (IL-12), that enhances the immune response (adjuvanteffect). It is understood that each of the components to be expressed isplaced under the control of elements required for expression in amammalian cell.

[0075] Consistent with the third aspect of the invention is acomposition comprising several vaccine vectors, each of them capable ofexpressing a polypeptide or derivative of the invention. A compositionmay also comprise a vaccine vector capable of expressing an additionalChlamydia antigen, or a subunit, fragment, homolog, mutant, orderivative thereof; optionally together with or a cytokine such as IL-2or IL-12.

[0076] Vaccination methods for treating or preventing infection in amammal comprises use of a vaccine vector of the invention to beadministered by any conventional route, particularly to a mucosal (e.g.,ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal,vaginal, or urinary tract) surface or via the parenteral (e.g.,subcutaneous, intradermal, intramuscular, intravenous, orintraperitoneal) route. Preferred routes depend upon the choice of thevaccine vector. Treatment may be effected in a single dose or repeatedat intervals. The appropriate dosage depends on various parametersunderstood by skilled artisans such as the vaccine vector itself, theroute of administration or the condition of the mammal to be vaccinated(weight, age and the like).

[0077] Live vaccine vectors available in the art include viral vectorssuch as adenoviruses and poxviruses as well as bacterial vectors, e.g.,Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié deCalmette-Guérin (BCG), and Streptococcus.

[0078] An example of an adenovirus vector, as well as a method forconstructing an adenovirus vector capable of expressing a DNA moleculeof the invention, are described in U.S. Pat. No. 4,920,209. Poxvirusvectors include vaccinia and canary pox virus, described in U.S. Pat.No. 4,722,848 and U.S. Pat. No. 5,364,773, respectively. (Also see,e.g., Tartaglia et al., Virology (1992) 188:217) for a description of avaccinia virus vector and Taylor et al, Vaccine (1995) 13:539 for areference of a canary pox.) Poxvirus vectors capable of expressing apolynucleotide of the invention are obtained by homologous recombinationas described in Kieny et al., Nature (1984) 312:163 so that thepolynucleotide of the invention is inserted in the viral genome underappropriate conditions for expression in mammalian cells. Generally, thedose of vaccine viral vector, for therapeutic or prophylactic use, canbe of from about 1×10⁴ to about 1×10¹¹, advantageously from about 1×10⁷to about 1×10¹, preferably of from about 1×10⁷ to about 1×10⁹plaque-forming units per kilogram. Preferably, viral vectors areadministered parenterally; for example, in 3 doses, 4 weeks apart. It ispreferable to avoid adding a chemical adjuvant to a compositioncontaining a viral vector of the invention and thereby minimizing theimmune response to the viral vector itself.

[0079] Non-toxicogenic Vibrio cholerae mutant strains that are useful asa live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551and U.S. Pat. No. 4,882,278 describe strains which have a substantialamount of the coding sequence of each of the two ctxA alleles deleted sothat no functional cholerae toxin is produced. WO 92/11354 describes astrain in which the irgA locus is inactivated by mutation; this mutationcan be combined in a single strain with ctxA mutations. WO 94/01533describes a deletion mutant lacking functional ctxA and attRS1 DNAsequences. These mutant strains are genetically engineered to expressheterologous antigens, as described in WO 94/19482. An effective vaccinedose of a Vibrio cholerae strain capable of expressing a polypeptide orpolypeptide derivative encoded by a DNA molecule of the inventioncontains about 1×10⁵ to about 1×10⁹, preferably about 1×10⁶ to about1×10⁸, viable bacteria in a volume appropriate for the selected route ofadministration. Preferred routes of administration include all mucosalroutes; most preferably, these vectors are administered intranasally ororally.

[0080] Attenuated Salmonella typhimurium strains, genetically engineeredfor recombinant expression of heterologous antigens or not, and theiruse as oral vaccines are described in Nakayama et al. (Bio/Technology(1988) 6:693) and WO 92/11361. Preferred routes of administrationinclude all mucosal routes; most preferably, these vectors areadministered intranasally or orally.

[0081] Other bacterial strains used as vaccine vectors in the context ofthe present invention are described for Shigella flexneri in High etal., EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299;for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci.USA (1995) 92:6868; and for Bacille Calmette Guerin in Flynn J.L., Cell.Mol. Biol. (1994) 40 (suppl. I):31, WO 88/06626, WO 90/00594, WO91/13157, WO 92/01796, and WO 92/21376.

[0082] In bacterial vectors, the polynucleotide of the invention isinserted into the bacterial genome or remains in a free state as part ofa plasmid.

[0083] The composition comprising a vaccine bacterial vector of thepresent invention may further contain an adjuvant. A number of adjuvantsare known to those skilled in the art. Preferred adjuvants are selectedas provided below.

[0084] Accordingly, a fourth aspect of the invention provides (i) acomposition of matter comprising a polynucleotide of the invention,together with a diluent or carrier; (ii) a pharmaceutical compositioncomprising a therapeutically or prophylactically effective amount of apolynucleotide of the invention; (iii) a method for inducing an immuneresponse against Chlamydia in a mammal by administration of animmunogenically effective amount of a polynucleotide of the invention toelicit a protective immune response to Chlamydia; and particularly, (iv)a method for preventing and/or treating a Chlamydia (e.g., C.trachomatis, C. psittaci, C. pneumoniae, or C. pecorum) infection, byadministering a prophylactic or therapeutic amount of a polynucleotideof the invention to an infected individual. Additionally, the fourthaspect of the invention encompasses the use of a polynucleotide of theinvention in the preparation of a medicament for preventing and/ortreating Chlamydia infection. A preferred use includes the use of a DNAmolecule placed under conditions for expression in a mammalian cell,especially in a plasmid that is unable to replicate in mammalian cellsand to substantially integrate in a mammalian genome.

[0085] Use of the polynucleotides of the invention include theiradministration to a mammal as a vaccine, for therapeutic or prophylacticpurposes. Such polynucleotides are used in the form of DNA as part of aplasmid that is unable to replicate in a mammalian cell and unable tointegrate into the mammalian genome. Typically, such a DNA molecule isplaced under the control of a promoter suitable for expression in amammalian cell. The promoter functions either ubiquitously ortissue-specifically. Examples of non-tissue specific promoters includethe early Cytomegalovirus (CMV) promoter (described in U.S. Pat. No.4,168,062) and the Rous Sarcoma Virus promoter (described in Norton &Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specificpromoter is the desmin promoter which drives expression in muscle cells(Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991)266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use ofpromoters is well-known to those skilled in the art. Useful vectors aredescribed in numerous publications, specifically WO 94/21797 andHartikka et al., Human Gene Therapy (1996) 7:1205.

[0086] Polynucleotides of the invention which are used as vaccinesencode either a precursor or a mature form of the correspondingpolypeptide. In the precursor form, the signal peptide is eitherhomologous or heterologous. In the latter case, a eucaryotic leadersequence such as the leader sequence of the tissue-type plasminogenfactor (tPA) is preferred.

[0087] As used herein, a composition of the invention contains one orseveral polynucleotides with optionally at least one additionalpolynucleotide encoding another Chlamydia antigen such as urease subunitA, B, or both, or a fragment, derivative, mutant, or analog thereof. Thecomposition may also contain an additional polynucleotide encoding acytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so thatthe immune response is enhanced. These additional polynucleotides areplaced under appropriate control for expression. Advantageously, DNAmolecules of the invention and/or additional DNA molecules to beincluded in the same composition, are present in the same plasmid.

[0088] Standard techniques of molecular biology for preparing andpurifying polynucleotides are used in the preparation of polynucleotidetherapeutics of the invention. For use as a vaccine, a polynucleotide ofthe invention is formulated according to various methods outlined below.

[0089] One method utililizes the polynucleotide in a naked form, free ofany delivery vehicles. Such a polynucleotide is simply diluted in aphysiologically acceptable solution such as sterile saline or sterilebuffered saline, with or without a carrier. When present, the carrierpreferably is isotonic, hypotonic, or weakly hypertonic, and has arelatively low ionic strength, such as provided by a sucrose solution,e.g., a solution containing 20% sucrose.

[0090] An alternative method utilizes the polynucleotide in associationwith agents that assist in cellular uptake. Examples of such agents are(i) chemicals that modify cellular permeability, such as bupivacaine(see, e.g., WO 94/16737), (ii) liposomes for encapsulation of thepolynucleotide, or (iii) cationic lipids or silica, gold, or tungstenmicroparticles which associate themselves with the polynucleotides.

[0091] Anionic and neutral liposomes are well-known in the art (see,e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), fora detailed description of methods for making liposomes) and are usefulfor delivering a large range of products, including polynucleotides.

[0092] Cationic lipids are also known in the art and are commonly usedfor gene delivery. Such lipids include Lipofectin T also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine), as described in WO 90/11092 as an example.

[0093] Formulation containing cationic liposomes may optionally containother transfection-facilitating compounds. A number of them aredescribed in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397.They include spermine derivatives useful for facilitating the transportof DNA through the nuclear membrane (see, for example, WO 93/18759) andmembrane-permeabilizing compounds such as GALA, Gramicidine S, andcationic bile salts (see, for example, WO 93/19768).

[0094] Gold or tungsten microparticles are used for gene delivery, asdescribed in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992)356:152. The microparticle-coated polynucleotide is injected viaintradermal or intraepidermal routes using a needleless injection device(“gene gun”), such as those described in U.S. Pat. No. 4,945,050, U.S.Pat. No. 5,015,580, and WO 94/24263.

[0095] The amount of DNA to be used in a vaccine recipient depends,e.g., on the strength of the promoter used in the DNA construct, theimmunogenicity of the expressed gene product, the condition of themammal intended for administration (e.g., the weight, age, and generalhealth of the mammal), the mode of administration, and the type offormulation. In general, a therapeutically or prophylactically effectivedose from about 1 μg to about 1 mg, preferably, from about 10 μg toabout 800 μg and, more preferably, from about 25 pg to about 250 pg, canbe administered to human adults. The administration can be achieved in asingle dose or repeated at intervals.

[0096] The route of administration is any conventional route used in thevaccine field. As general guidance, a polynucleotide of the invention isadministered via a mucosal surface, e.g., an ocular, intranasal,pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface;or via a parenteral route, e.g., by an intravenous, subcutaneous,intraperitoneal, intradermal, intraepidermal, or intramuscular route.The choice of administration route depends on the formulation that isselected. A polynucleotide formulated in association with bupivacaine isadvantageously administered into muscles. When a neutral or anionicliposome or a cationic lipid, such as DOTMA or DC-Chol, is used, theformulation can be advantageously injected via intravenous, intranasal(aerosolization), intramuscular, intradermal, and subcutaneous routes. Apolynucleotide in a naked form can advantageously be administered viathe intramuscular, intradermal, or subcutaneous routes.

[0097] Although not absolutely required, such a composition can alsocontain an adjuvant. If so, a systemic adjuvant that does not requireconcomitant administration in order to exhibit an adjuvant effect ispreferable such as, e.g., QS21, which is described in U.S. Pat. No.5,057,546.

[0098] The sequence information provided in the present applicationenables the design of specific nucleotide probes and primers that areused for diagnostic purposes. Accordingly, a fifth aspect of theinvention provides a nucleotide probe or primer having a sequence foundin or derived by degeneracy of the genetic code from a sequence shown inSEQ ID No:1.

[0099] The term “probe” as used in the present application refers to DNA(preferably single stranded) or RNA molecules (or modifications orcombinations thereof) that hybridize under the stringent conditions, asdefined above, to nucleic acid molecules having SEQ ID No:1 or tosequences homologous to SEQ ID No:1, or to its complementary oranti-sense sequence. Generally, probes are significantly shorter thanfull-length sequences. Such probes contain from about 5 to about 100,preferably from about 10 to about 80, nucleotides. In particular, probeshave sequences that are at least 75%, preferably at least 85%, morepreferably 95% homologous to a portion of SEQ ID No:1 or that arecomplementary to such sequences. Probes may contain modified bases suchas inosine, methyl-5-deoxycytidine, deoxyuridine,dimethylamino-5-deoxyuridine, or diamino-2, 6-purine. Sugar or phosphateresidues may also be modified or substituted. For example, a deoxyriboseresidue may be replaced by a polyamide (Nielsen et al., Science (1991)254:1497) and phosphate residues may be replaced by ester groups such asdiphosphate, alkyl, arylphosphonate and phosphorothioate esters. Inaddition, the 2′-hydroxyl group on ribonucleotides may be modified byincluding such groups as alkyl groups.

[0100] Probes of the invention are used in diagnostic tests, as captureor detection probes. Such capture probes are conventionally immobilizedon a solid support, directly or indirectly, by covalent means or bypassive adsorption. A detection probe is labelled by a detection markerselected from: radioactive isotopes, enzymes such as peroxidase,alkaline phosphatase, and enzymes able to hydrolyze a chromogenic,fluorogenic, or luminescent substrate, compounds that are chromogenic,fluorogenic, or luminescent, nucleotide base analogs, and biotin.

[0101] Probes of the invention are used in any conventionalhybridization technique, such as dot blot (Maniatis et al., MolecularCloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975)98:503), northern blot (identical to Southern blot with the exceptionthat RNA is used as a target), or the sandwich technique (Dunn et al.,Cell (1977) 12:23). The latter technique involves the use of a specificcapture probe and/or a specific detection probe with nucleotidesequences that at least partially differ from each other.

[0102] A primer is a probe of usually about 10 to about 40 nucleotidesthat is used to initiate enzymatic polymerization of DNA in anamplification process (e.g., PCR), in an elongation process, or in areverse transcription method. Primers used in diagnostic methodsinvolving PCR are labeled by methods known in the art.

[0103] As described herein, the invention also encompasses (i) a reagentcomprising a probe of the invention for detecting and/or identifying thepresence of Chlamydia in a biological material; (ii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA or RNA is extracted from the material anddenatured, and (c) exposed to a probe of the invention, for example, acapture, detection probe or both, under stringent hybridizationconditions, such that hybridization is detected; and (iii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA is extracted therefrom, (c) the extractedDNA is primed with at least one, and preferably two, primers of theinvention and amplified by polymerase chain reaction, and (d) theamplified DNA fragment is produced.

[0104] It is apparent that disclosure of polynucleotide sequences of SEQID No:1, its homologs and partial sequences enable their correspondingamino acid sequences. Accordingly, a sixth aspect of the inventionfeatures a substantially purified polypeptide or polypeptide derivativehaving an amino acid sequence encoded by a polynucleotide of theinvention.

[0105] A “substantially purified polypeptide” as used herein is definedas a polypeptide that is separated from the environment in which itnaturally occurs and/or that is free of the majority of the polypeptidesthat are present in the environment in which it was synthesized. Forexample, a substantially purified polypeptide is free from cytoplasmicpolypeptides. Those skilled in the art would readily understand that thepolypeptides of the invention may be purified from a natural source,i.e., a Chlamydia strain, or produced by recombinant means.

[0106] Consistent with the sixth aspect of the invention arepolypeptides, homologs or fragments which are modified or treated toenhance their immunogenicity in the target animal, in whom thepolypeptide, homolog or fragments are intended to confer protectionagainst Chlamydia. Such modifications or treatments include: amino acidsubstitutions with an amino acid derivative such as 3-methyhistidine,4-hydroxyproline, 5-hydroxylysine etc., modifications or deletions whichare carried out after preparation of the polypeptide, homolog orfragment, such as the modification of free amino, carboxyl or hydroxylside groups of the amino acids.

[0107] Identification of homologous polypeptides or polypeptidederivatives encoded by polynucleotides of the invention which havespecific antigenicity is achieved by screening for cross-reactivity withan antiserum raised against the polypeptide of reference having an aminoacid sequence of SEQ ID No:1. The procedure is as follows: amonospecific hyperimmune antiserum is raised against a purifiedreference polypeptide, a fusion polypeptide (for example, an expressionproduct of MBP, GST, or His-tag systems, the description andinstructions for use of which are contained in Invitrogen productmanuals for pcDNA3.1/Myc-His(+) A, B, and C and for the Xpress™ SystemProtein Purification), or a synthetic peptide predicted to be antigenic.Where an antiserum is raised against a fusion polypeptide, two differentfusion systems are employed. Specific antigenicity can be determinedaccording to a number of methods, including Western blot (Towbin et al.,Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, asdescribed below.

[0108] In a Western blot assay, the product to be screened, either as apurified preparation or a total E. coli extract, is submitted toSDS-Page electrophoresis as described by Laemmli (Nature (1970)227:680). After transfer to a nitrocellulose membrane, the material isfurther incubated with the monospecific hyperimmune antiserum diluted inthe range of dilutions from about 1:5 to about 1:5000, preferably fromabout 1:100 to about 1:500. Specific antigenicity is shown once a bandcorresponding to the product exhibits reactivity at any of the dilutionsin the above range.

[0109] In an ELISA assay, the product to be screened is preferably usedas the coating antigen. A purified preparation is preferred, although awhole cell extract can also be used. Briefly, about 100 pl of apreparation at about 10 pg protein/ml are distributed into wells of a96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at37° C. then overnight at 4° C. The plate is washed with phosphate buffersaline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells aresaturated with 250 pl PBS containing 1% bovine serum albumin (BSA) toprevent non-specific antibody binding. After 1 hour incubation at 37°C., the plate is washed with PBS/Tween buffer. The antiserum is seriallydiluted in PBS/Tween buffer containing 0.5% BSA. 100 pl of dilutions areadded per well. The plate is incubated for 90 minutes at 37° C., washedand evaluated according to standard procedures. For example, a goatanti-rabbit peroxidase conjugate is added to the wells when specificantibodies were raised in rabbits. Incubation is carried out for 90minutes at 37° C. and the plate is washed. The reaction is developedwith the appropriate substrate and the reaction is measured bycolorimetry (absorbance measured spectrophotometrically). Under theabove experimental conditions, a positive reaction is shown by O.D.values greater than a non immune control serum.

[0110] In a dot blot assay, a purified product is preferred, although awhole cell extract can also be used. Briefly, a solution of the productat about 100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH7.5). 100 pl of each dilution are applied to a nitrocellulose membrane0.45 pm set in a 96-well dot blot apparatus (Biorad). The buffer isremoved by applying vacuum to the system. Wells are washed by additionof 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membraneis saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10g/L skim milk) and incubated with an antiserum dilution from about 1:50to about 1:5000, preferably about 1:500. The reaction is revealedaccording to standard procedures. For example, a goat anti-rabbitperoxidase conjugate is added to the wells when rabbit antibodies areused. Incubation is carried out 90 minutes at 37° C. and the blot iswashed. The reaction is developed with the appropriate substrate andstopped. The reaction is measured visually by the appearance of acolored spot, e.g., by colorimetry. Under the above experimentalconditions, a positive reaction is shown once a colored spot isassociated with a dilution of at least about 1:5, preferably of at leastabout 1:500.

[0111] Therapeutic or prophylactic efficacy of a polypeptide orderivative of the invention can be evaluated as described below. Aseventh aspect of the invention provides (i) a composition of mattercomprising a polypeptide of the invention together with a diluent orcarrier; specifically (ii) a pharmaceutical composition containing atherapeutically or prophylactically effective amount of a polypeptide ofthe invention; (iii) a method for inducing an immune response againstChlamydia in a mammal, by administering to the mammal an immunogenicallyeffective amount of a polypeptide of the invention to elicit aprotective immune response to Chlamydia; and particularly, (iv) a methodfor preventing and/or treating a Chlamydia (e.g., C. trachomatis. C.psittaci, C. pneumoniae. or C. pecorum) infection, by administering aprophylactic or therapeutic amount of a polypeptide of the invention toan infected individual. Additionally, the seventh aspect of theinvention encompasses the use of a polypeptide of the invention in thepreparation of a medicament for preventing and/or treating Chlamydiainfection.

[0112] As used herein, the immunogenic compositions of the invention areadministered by conventional routes known the vaccine field, inparticular to a mucosal (e.g., ocular, intranasal, pulmonary, oral,gastric, intestinal, rectal, vaginal, or urinary tract) surface or viathe parenteral (e.g., subcutaneous, intradermal, intramuscular,intravenous, or intraperitoneal) route. The choice of administrationroute depends upon a number of parameters, such as the adjuvantassociated with the polypeptide. If a mucosal adjuvant is used, theintranasal or oral route is preferred. If a lipid formulation or analuminum compound is used, the parenteral route is preferred with thesub-cutaneous or intramuscular route being most preferred. The choicealso depends upon the nature of the vaccine agent. For example, apolypeptide of the invention fused to CTB or LTB is best administered toa mucosal surface.

[0113] As used herein, the composition of the invention contains one orseveral polypeptides or derivatives of the invention. The compositionoptionally contains at least one additional Chlamydia antigen, or asubunit, fragment, homolog, mutant, or derivative thereof.

[0114] For use in a composition of the invention, a polypeptide orderivative thereof is formulated into or with liposomes, preferablyneutral or anionic liposomes, microspheres, ISCOMS, orvirus-like-particles (VLPS) to facilitate delivery and/or enhance theimmune response. These compounds are readily available to one skilled inthe art; for example, see Liposomes: A Practical Approach, RCP New Ed,IRL press (1990).

[0115] Adjuvants other than liposomes and the like are also used and areknown in the art. Adjuvants may protect the antigen from rapid dispersalby sequestering it in a local deposit, or they may contain substancesthat stimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. An appropriateselection can conventionally be made by those skilled in the art, forexample, from those described below (under the eleventh aspect of theinvention).

[0116] Treatment is achieved in a single dose or repeated as necessaryat intervals, as can be determined readily by one skilled in the art.For example, a priming dose is followed by three booster doses at weeklyor monthly intervals. An appropriate dose depends on various parametersincluding the recipient (e.g., adult or infant), the particular vaccineantigen, the route and frequency of administration, the presence/absenceor type of adjuvant, and the desired effect (e.g., protection and/ortreatment), as can be determined by one skilled in the art. In general,a vaccine antigen of the invention is administered by a mucosal route inan amount from about 10 μg to about 500 mg, preferably from about 1 mgto about 200 mg. For the parenteral route of administration, the doseusually does not exceed about 1 mg, preferably about 100 μg.

[0117] When used as vaccine agents, polynucleotides and polypeptides ofthe invention may be used sequentially as part of a multistepimmunization process. For example, a mammal is initially primed with avaccine vector of the invention such as a pox virus, e.g., via theparenteral route, and then boosted twice with the polypeptide encoded bythe vaccine vector, e.g., via the mucosal route. In another example,liposomes associated with a polypeptide or derivative of the inventionis also used for priming, with boosting being carried out mucosallyusing a soluble polypeptide or derivative of the invention incombination with a mucosal adjuvant (e.g., LT).

[0118] A polypeptide derivative of the invention is also used inaccordance with the seventh aspect as a diagnostic reagent for detectingthe presence of anti-Chlamydia antibodies, e.g., in a blood sample. Suchpolypeptides are about 5 to about 80, preferably about 10 to about 50amino acids in length. They are either labeled or unlabeled, dependingupon the diagnostic method. Diagnostic methods involving such a reagentare described below.

[0119] Upon expression of a DNA molecule of the invention, a polypeptideor polypeptide derivative is produced and purified using knownlaboratory techniques. As described above, the polypeptide orpolypeptide derivative may be produced as a fusion protein containing afused tail that facilitates purification. The fusion product is used toimmunize a small mammal, e.g., a mouse or a rabbit, in order to raiseantibodies against the polypeptide or polypeptide derivative(monospecific antibodies). Accordingly, an eighth aspect of theinvention provides a monospecific antibody that binds to a polypeptideor polypeptide derivative of the invention.

[0120] By “monospecific antibody” is meant an antibody that is capableof reacting with a unique naturally-occurring Chlamydia polypeptide. Anantibody of the invention is either polyclonal or monoclonal.Monospecific antibodies may be recombinant, e.g., chimeric (e.g.,constituted by a variable region of murine origin associated with ahuman constant region), humanized (a human immunoglobulin constantbackbone together with hypervariable region of animal, e.g., murine,origin), and/or single chain. Both polyclonal and monospecificantibodies may also be in the form of immunoglobulin fragments, e.g.,F(ab)′2 or Fab fragments. The antibodies of the invention are of anyisotype, e.g., IgG or IgA, and polyclonal antibodies are of a singleisotype or a mixture of isotypes.

[0121] Antibodies against the polypeptides, homologs or fragments of thepresent invention are generated by immunization of a mammal with acomposition comprising said polypeptide, homolog or fragment. Suchantibodies may be polyclonal or monoclonal. Methods to producepolyclonal or monoclonal antibodies are well known in the art. For areview, see “Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al.,1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, seeKohler & Milstein (1975) Nature 256:495-497.

[0122] The antibodies of the invention, which are raised to apolypeptide or polypeptide derivative of the invention, are produced andidentified using standard immunological assays, e.g., Western blotanalysis, dot blot assay, or ELISA (see, e.g., Coligan et al., CurrentProtocols in Immunology (1994) John Wiley & Sons, Inc., New York, N.Y.).The antibodies are used in diagnostic methods to detect the presence ofa Chlamydia antigen in a sample, such as a biological sample. Theantibodies are also used in affinity chromatography for purifying apolypeptide or polypeptide derivative of the invention. As is discussedfurther below, such antibodies may be used in prophylactic andtherapeutic passive immunization methods.

[0123] Accordingly, a ninth aspect of the invention provides (i) areagent for detecting the presence of Chlamydia in a biological samplethat contains an antibody, polypeptide, or polypeptide derivative of theinvention; and (ii) a diagnostic method for detecting the presence ofChlamydia in a biological sample, by contacting the biological samplewith an antibody, a polypeptide, or a polypeptide derivative of theinvention, such that an immune complex is formed, and by detecting suchcomplex to indicate the presence of Chlamydia in the sample or theorganism from which the sample is derived.

[0124] Those skilled in the art will readily understand that the immunecomplex is formed between a component of the sample and the antibody,polypeptide, or polypeptide derivative, whichever is used, and that anyunbound material is removed prior to detecting the complex. It isunderstood that a polypeptide reagent is useful for detecting thepresence of anti-Chlamydia antibodies in a sample, e.g., a blood sample,while an antibody of the invention is used for screening a sample, suchas a gastric extract or biopsy, for the presence of Chlamydiapolypeptides.

[0125] For diagnostic applications, the reagent (i.e., the antibody,polypeptide, or polypeptide derivative of the invention) is either in afree state or immobilized on a solid support, such as a tube, a bead, orany other conventional support used in the field. Immobilization isachieved using direct or indirect means. Direct means include passiveadsorption (non-covalent binding) or covalent binding between thesupport and the reagent. By “indirect means” is meant that ananti-reagent compound that interacts with a reagent is first attached tothe solid support. For example, if a polypeptide reagent is used, anantibody that binds to it can serve as an anti-reagent, provided that itbinds to an epitope that is not involved in the recognition ofantibodies in biological samples. Indirect means may also employ aligand-receptor system, for example, where a molecule such as a vitaminis grafted onto the polypeptide reagent and the corresponding receptorimmobilized on the solid phase. This is illustrated by thebiotin-streptavidin system. Alternatively, a peptide tail is addedchemically or by genetic engineering to the reagent and the grafted orfused product immobilized by passive adsorption or covalent linkage ofthe peptide tail.

[0126] Such diagnostic agents may be included in a kit which alsocomprises instructions for use. The reagent is labeled with a detectionmeans which allows for the detection of the reagent when it is bound toits target. The detection means may be a fluorescent agent such asfluorescein isocyanate or fluorescein isothiocyanate, or an enzyme suchas horse radish peroxidase or luciferase or alkaline phosphatase, or aradioactive element such as ¹²⁵I or ⁵¹Cr.

[0127] Accordingly, a tenth aspect of the invention provides a processfor purifying, from a biological sample, a polypeptide or polypeptidederivative of the invention, which involves carrying out antibody-basedaffinity chromatography with the biological sample, wherein the antibodyis a monospecific antibody of the invention.

[0128] For use in a purification process of the invention, the antibodyis either polyclonal or monospecific, and preferably is of the IgG type.Purified IgGs is prepared from an antiserum using standard methods (see,e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley& Sons, Inc., New York, N.Y.). Conventional chromatography supports, aswell as standard methods for grafting antibodies, are described in,e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988)and outlined below.

[0129] Briefly, a biological sample, such as an C. pneumoniae extractpreferably in a buffer solution, is applied to a chromatographymaterial, preferably equilibrated with the buffer used to dilute thebiological sample so that the polypeptide or polypeptide derivative ofthe invention (i.e., the antigen) is allowed to adsorb onto thematerial. The chromatography material, such as a gel or a resin coupledto an antibody of the invention, is in either a batch form or a column.The unbound components are washed off and the antigen is then elutedwith an appropriate elution buffer, such as a glycine buffer or a buffercontaining a chaotropic agent, e.g., guanidine HCl, or high saltconcentration (e.g., 3 M MgCl₂). Eluted fractions are recovered and thepresence of the antigen is detected, e.g., by measuring the absorbanceat 280 nm.

[0130] An eleventh aspect of the invention provides (i) a composition ofmatter comprising a monospecific antibody of the invention, togetherwith a diluent or carrier; (ii) a pharmaceutical composition comprisinga therapeutically or prophylactically effective amount of a monospecificantibody of the invention, and (iii) a method for treating or preventinga Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C.pecorum) infection, by administering a therapeutic or prophylacticamount of a monospecific antibody of the invention to an infectedindividual. Additionally, the eleventh aspect of the inventionencompasses the use of a monospecific antibody of the invention in thepreparation of a medicament for treating or preventing Chlamydiainfection.

[0131] The monospecific antibody is either polyclonal or monoclonal,preferably of the IgA isotype (predominantly). In passive immunization,the antibody is administered to a mucosal surface of a mammal, e.g., thegastric mucosa, e.g., orally or intragastrically, advantageously, in thepresence of a bicarbonate buffer. Alternatively, systemicadministration, not requiring a bicarbonate buffer, is carried out. Amonospecific antibody of the invention is administered as a singleactive component or as a mixture with at least one monospecific antibodyspecific for a different Chlamydia polypeptide. The amount of antibodyand the particular regimen used are readily determined by one skilled inthe art. For example, daily administration of about 100 to 1,000 mg ofantibodies over one week, or three doses per day of about 100 to 1,000mg of antibodies over two or three days, are effective regimens for mostpurposes.

[0132] Therapeutic or prophylactic efficacy are evaluated using standardmethods in the art, e.g., by measuring induction of a mucosal immuneresponse or induction of protective and/or therapeutic immunity, using,e.g., the C. pneumoniae mouse model. Those skilled in the art willreadily recognize that the C. pneumoniae strain of the model may bereplaced with another Chlamydia strain. For example, the efficacy of DNAmolecules and polypeptides from C. pneumoniae is preferably evaluated ina mouse model using C. pneumoniae strain. Protection is determined bycomparing the degree of Chlamydia infection to that of a control group.Protection is shown when infection is reduced by comparison to thecontrol group. Such an evaluation is made for polynucleotides, vaccinevectors, polypeptides and derivatives thereof, as well as antibodies ofthe invention.

[0133] Adjuvants useful in any of the vaccine compositions describedabove are as follows.

[0134] Adjuvants for parenteral administration include aluminumcompounds, such as aluminum hydroxide, aluminum phosphate, and aluminumhydroxy phosphate. The antigen is precipitated with, or adsorbed onto,the aluminum compound according to standard protocols. Other adjuvants,such as RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteraladministration.

[0135] Adjuvants for mucosal administration include bacterial toxins,e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), theClostridium difficile toxin A and the pertussis toxin (PT), orcombinations, subunits, toxoids, or mutants thereof such as a purifiedpreparation of native cholera toxin subunit B (CTB). Fragments,homologs, derivatives, and fusions to any of these toxins are alsosuitable, provided that they retain adjuvant activity. Preferably, amutant having reduced toxicity is used. Suitable mutants are described,e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LTmutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).Additional LT mutants that are used in the methods and compositions ofthe invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, andGlu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryllipid A (MPLA) of, e.g., E. coli, Salmonella Minnesota, Salmonellatyphimurium, or Shigella flexneri; saponins, or polylactide glycolide(PLGA) microspheres, is also be used in mucosal administration.

[0136] Adjuvants useful for both mucosal and parenteral administrationsinclude polyphosphazene (WO 95/02415), DC-chol (3b-(N-(N′,N′-dimethylaminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and WO96/14831) and QS-21 (WO 88/09336).

[0137] Any pharmaceutical composition of the invention containing apolynucleotide, a polypeptide, a polypeptide derivative, or an antibodyof the invention, is manufactured in a conventional manner. Inparticular, it is formulated with a pharmaceutically acceptable diluentor carrier, e.g., water or a saline solution such as phosphate buffersaline. In general, a diluent or carrier is selected on the basis of themode and route of administration, and standard pharmaceutical practice.Suitable pharmaceutical carriers or diluents, as well as pharmaceuticalnecessities for their use in pharmaceutical formulations, are describedin Remington's Pharmaceutical Sciences, a standard reference text inthis field and in the USP/NF.

[0138] The invention also includes methods in which Chlamydia infectionare treated by oral administration of a Chlamydia polypeptide of theinvention and a mucosal adjuvant, in combination with an antibiotic, anantacid, sucralfate, or a combination thereof. Examples of suchcompounds that can be administered with the vaccine antigen and theadjuvant are antibiotics, including, e.g., macrolides, tetracyclines,and derivatives thereof (specific examples of antibiotics that can beused include azithromycin or doxicyclin or immunomodulators such ascytokines or steroids). In addition, compounds containing more than oneof the above-listed components coupled together, are used. The inventionalso includes compositions for carrying out these methods, i.e.,compositions containing a Chlamydia antigen (or antigens) of theinvention, an adjuvant, and one or more of the above-listed compounds,in a pharmaceutically acceptable carrier or diluent.

[0139] It has recently been shown that the 60kDa cysteine rich membraneprotein contains a sequence cross-reactive with the murine alpha-myosinheavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaieret al., Science (1999) 283:1335). This cross-reactivity is proposed tocontribute to the development of cardiovascular disease, so it may bebeneficial to remove this epitope, and any other epitopes cross-reactivewith human antigens, from the protein if it is to be used as a vaccine.Accordingly, a further embodiment of the present invention includes themodification of the coding sequence, for example, by deletion orsubstitution of the nucleotides encoding the epitope frompolynucleotides encoding the protein, as to improve the efficacy andsafety of the protein as a vaccine. A similar approach may beappropriate for any protective antigen found to have unwanted homologiesor cross-reactivities with human antigens.

[0140] Amounts of the above-listed compounds used in the methods andcompositions of the invention are readily determined by one skilled inthe art. Treatment/immunization schedules are also known and readilydesigned by one skilled in the art. For example, the non-vaccinecomponents can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28.

EXAMPLES

[0141] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples. These examples are described solely forpurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitation.

Example 1

[0142] This example illustrates the preparation of plasmid vectorpCABk831 containing the omp P6 precursor gene.

[0143] The omp P6 precursor gene was amplified from Chlamydia pneumoniaegenomic DNA by polymerase chain reaction (PCR) using a 5′ primer (5′ATAAGAATGCGGCCGCCACCATGAATATACATTCCCTATGGAAAC 3′; SEQ ID No:3) and a 3′primer (5′ GCGCCGGATCCCGCGTGCATGAATCTTAAACTCTGTACGGC 3′; SEQ ID No:4).

[0144] The 5′ primer contains a NotI restriction site, a ribosomebinding site, an initiation codon and a sequence at the 5′ end of theomp P6 precursor coding sequence. The 3′ primer includes the sequenceencoding the C-terminal sequence of the omp P6 precursor gene and aBamHI restriction site. The stop codon was excluded and an additionalnucleotide was inserted to obtain an in-frame fusion with the Histidinetag.

[0145] After amplification, the PCR fragment was purified usingQIAquick” PCR purification kit (Qiagen), digested with NotI and BamHIand cloned into the pCA-Myc-His eukaryotic expression vector describedin Example 2 (FIG. 3) with transcription under control of the human CMVpromoter.

Example 2

[0146] This example illustrates the preparation of the eukaryoticexpression vector pCA/Myc-His.

[0147] Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted withSpeI and BamHI to remove the CMV promoter and the remaining vectorfragment was isolated. The CMV promoter and intron A from plasmidVR-1012 (Vical) was isolated on a SpeI/BamHI fragment. The fragmentswere ligated together to produce plasmid pCA/Myc-His. The NotI/BamHIrestricted PCR fragment containing the omp P6 precursor gene was ligatedinto the NotI and BamHI restricted plasmid pCA/Myc-His to produceplasmid pCABk831 (FIG. 3).

[0148] The resulting plasmid, pCABk831, was transferred byelectroporation into E. coli XL-1 blue (Stratagene) which was grown inLB broth containing 50 μg/ml carbenicillin. The plasmid was isolated bythe Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purificationsystem. DNA concentration was determined by absorbance at 260 nm and theplasmid was verified after gel electrophoresis and ethidium bromidestaining by comparison to molecular weight standards. The 5′ and 3′ endsof the gene were verified by sequencing using a LiCor model 4000 L DNAsequencer and IRD-800 labelled primers.

Example 3

[0149] This example illustrates the immunization of mice to achieveprotection against an intranasal challenge of C. pneumoniae.

[0150] It has been previously demonstrated (Yang et al. Infect. Immun.May 1993. 61(5):2037-40) that mice are susceptible to intranasalinfection with different isolates of C. pneumoniae. Strain AR-39(Grayston et al (1990) Journal of Infectious Diseases 161:618-625) wasused in Balb/c mice as a challenge infection model to examine thecapacity of Chlamydia gene products delivered as naked DNA to elicit aprotective response against a sublethal C. pneumoniae lung infection.Protective immunity is defined as an accelerated clearance of pulmonaryinfection.

[0151] Groups of 7 to 9 week old male Balb/c mice (8 to 10 per group)were immunized intramuscularly (i.m.) plus intranasally (i.n.) withplasmid DNA containing the C. pneumoniae omp P6 precursor gene asdescribed in Examples 1 and 2. Saline or the plasmid vector lacking aninserted Chlamydial gene was given to groups of control animals.

[0152] For i.m. immunization, alternate left and right quadriceps wereinjected with 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3and 6 weeks. For i.n. immunization, anaesthetized mice were aspirated 50μl of PBS containing 50 μg DNA on three occasions at 0, 3 and 6 weeks.At week 8, immunized mice were inoculated i.n. with 5×10⁵ IFU of C.pneumoniae, strain AR39 in 100 μl of SPG buffer to test their ability tolimit the growth of a sublethal C. pneumoniae challenge.

[0153] Lungs were taken from mice at day 9 post-challenge andimmediately homogenised in SPG buffer (7.5% sucrose, 5 mM glutamate,12.5 mM phosphate pH 7.5). The homogenate was stored frozen at −70° C.until assay. Dilutions of the homogenate were assayed for the presenceof infectious Chlamydia by inoculation onto monolayers of susceptiblecells. The inoculum was centrifuged onto the cells at 3000 rpm for 1hour, then the cells were incubated for three days at 35° C. in thepresence of 1 μg/ml cycloheximide. After incubation the monolayers werefixed with formalin and methanol then immunoperoxidase stained for thepresence of Chlamydial inclusions using convalescent sera from rabbitsinfected with C. pneumoniae and metal-enhanced DAB as a peroxidasesubstrate.

[0154]FIG. 4 and Table 1 show that mice immunized i.n. and i.m. withpCABk831 had Chlamydial lung titers less than 39,500 in 5 of 6 cases atday 9 (mean 24,933) whereas the range of values for control mice shamimmunized with saline was 13,500 to 178,700 IFU/lung (mean 69,782) atday 9. DNA immunisation per se was not responsible for the observedprotective effect since another plasmid DNA construct, pCABk1106, failedto protect, with lung titers in immunised mice similar to those obtainedfor saline-immunized control mice (mean 62,516). The construct pCABk1106is identical to pCABk831 except that the nucleotide sequence encodingthe putative omp P6 precursor is replaced with a C. pneumoniaenucleotide sequence encoding an unrelated hypothetical protein. TABLE 1BACTERIAL LOAD (INCLUSION FORMING UNITS PER LUNG) IN THE LUNGS OF BALB/CMICE IMMUNIZED WITH VARIOUS DNA IMMUNIZATION CONSTRUCTS IMMUNIZINGCONSTRUCT Saline pCABk1106 pCABk831 MOUSE Day 9 Day 9 Day 9 1 17300137900 39100 2 88800 43700 68200 3 90000 105200 3300 4 44500 45800 118005 156500 27600 8300 6 69700 14900 18900 7 13500 8 14100 9 71700 10103000 11 112100 12 59500 13 90200 14 178700 15 55400 16 23800 17 6670018 66800 19 53800 20 38700 21 127000 22 28000 23 35200 24 78900 MEAN69782.61 62516.67 24933.33 SD 44216.7 48217.32 24601.84 Wilcoxon 0.65940.0119 p

[0155]

1 4 1 779 DNA Chlamydia pneumoniae CDS (101)..(676) 1 gtctagtcaccaaaaaaact aacaaaattg ctataggagt aggagaaaaa cggttcccct 60 cctggggtgctttccctcag caaccgataa agagaacact atg aat ata cat tcc 115 Met Asn Ile HisSer 1 5 cta tgg aaa ctt tgt act tta ttg gct tta ctt gca ttg cca gca tgt163 Leu Trp Lys Leu Cys Thr Leu Leu Ala Leu Leu Ala Leu Pro Ala Cys 1015 20 agc ctt tcc cct aat tat ggc tgg gag gat tcc tgt aat aca tgc cat211 Ser Leu Ser Pro Asn Tyr Gly Trp Glu Asp Ser Cys Asn Thr Cys His 2530 35 cat aca aga cga aaa aag cct tct tct ttt ggc ttt gtt cct ctc tat259 His Thr Arg Arg Lys Lys Pro Ser Ser Phe Gly Phe Val Pro Leu Tyr 4045 50 acc gaa gag gac ttt aac cct aat ttt acc ttc ggt gag tat gat tcc307 Thr Glu Glu Asp Phe Asn Pro Asn Phe Thr Phe Gly Glu Tyr Asp Ser 5560 65 aaa gaa gaa aaa caa tac aag tca agc caa gtt gca gca ttt cgt aat355 Lys Glu Glu Lys Gln Tyr Lys Ser Ser Gln Val Ala Ala Phe Arg Asn 7075 80 85 atc acc ttt gct aca gac agc tat aca att aaa ggt gaa gag aac ctt403 Ile Thr Phe Ala Thr Asp Ser Tyr Thr Ile Lys Gly Glu Glu Asn Leu 9095 100 gcg att ctc acg aac ttg gtt cac tac atg aag aaa aac ccg aaa gct451 Ala Ile Leu Thr Asn Leu Val His Tyr Met Lys Lys Asn Pro Lys Ala 105110 115 aca ctg tac att gaa ggg cat act gac gag cgt gga gct gca tcc tat499 Thr Leu Tyr Ile Glu Gly His Thr Asp Glu Arg Gly Ala Ala Ser Tyr 120125 130 aac ctt gct tta gga gca cga cga gcc aat gcg att aaa gag cat ctc547 Asn Leu Ala Leu Gly Ala Arg Arg Ala Asn Ala Ile Lys Glu His Leu 135140 145 cga aag cag gga atc tct gca gat cgt cta tct act att tcc tac gga595 Arg Lys Gln Gly Ile Ser Ala Asp Arg Leu Ser Thr Ile Ser Tyr Gly 150155 160 165 aaa gaa cat cct tta aat tcg gga cac aac gaa cta gca tgg caacaa 643 Lys Glu His Pro Leu Asn Ser Gly His Asn Glu Leu Ala Trp Gln Gln170 175 180 aat cgc cgt aca gag ttt aag att cat gca cgc taagctaagcttttttattc 696 Asn Arg Arg Thr Glu Phe Lys Ile His Ala Arg 185 190ttctaagtct gttattttca ggaatagatt gcagtcgttt gcatgctgct ggaaggtctc 756cttctctaca aggagtcctt gca 779 2 192 PRT Chlamydia pneumoniae 2 Met AsnIle His Ser Leu Trp Lys Leu Cys Thr Leu Leu Ala Leu Leu 1 5 10 15 AlaLeu Pro Ala Cys Ser Leu Ser Pro Asn Tyr Gly Trp Glu Asp Ser 20 25 30 CysAsn Thr Cys His His Thr Arg Arg Lys Lys Pro Ser Ser Phe Gly 35 40 45 PheVal Pro Leu Tyr Thr Glu Glu Asp Phe Asn Pro Asn Phe Thr Phe 50 55 60 GlyGlu Tyr Asp Ser Lys Glu Glu Lys Gln Tyr Lys Ser Ser Gln Val 65 70 75 80Ala Ala Phe Arg Asn Ile Thr Phe Ala Thr Asp Ser Tyr Thr Ile Lys 85 90 95Gly Glu Glu Asn Leu Ala Ile Leu Thr Asn Leu Val His Tyr Met Lys 100 105110 Lys Asn Pro Lys Ala Thr Leu Tyr Ile Glu Gly His Thr Asp Glu Arg 115120 125 Gly Ala Ala Ser Tyr Asn Leu Ala Leu Gly Ala Arg Arg Ala Asn Ala130 135 140 Ile Lys Glu His Leu Arg Lys Gln Gly Ile Ser Ala Asp Arg LeuSer 145 150 155 160 Thr Ile Ser Tyr Gly Lys Glu His Pro Leu Asn Ser GlyHis Asn Glu 165 170 175 Leu Ala Trp Gln Gln Asn Arg Arg Thr Glu Phe LysIle His Ala Arg 180 185 190 3 45 DNA Artificial Sequence 5′ PCR primer 3ataagaatgc ggccgccacc atgaatatac attccctatg gaaac 45 4 41 DNA ArtificialSequence 3′ PCR primer 4 gcgccggatc ccgcgtgcat gaatcttaaa ctctgtacgg c41

1. A nucleic acid molecule comprising a nucleic acid sequence whichencodes a polypeptide selected from any one of: (a) SEQ ID No: 2; (b) animmunogenic fragment comprising at least 12 consecutive amino acids froma polypeptide of (a); and (c) a polypeptide of (a) or (b) which has beenmodified to improve its immunogenicity, wherein said modifiedpolypeptide is at least 75% identical in amino acid sequence to thecorresponding polypeptide of (a) or (b).
 2. A nucleic acid moleculecomprising a nucleic acid sequence selected from any one of: (a) SEQ IDNos: 1; (b) a sequence which encodes a polypeptide encoded by SEQ ID No:1; (c) a sequence comprising at least 38 consecutive nucleotides fromany one of the nucleic acid sequences of (a) and (b); and (d) a sequencewhich encodes a polypeptide which is at least 75% identical in aminoacid sequence to the polypeptides encoded by SEQ ID No:
 1. 3. A nucleicacid molecule comprising a nucleic acid sequence which is anti-sense tothe nucleic acid molecule of claim
 1. 4. A nucleic acid moleculecomprising a nucleic acid sequence which encodes a fusion protein, saidfusion protein comprising a polypeptide encoded by a nucleic acidmolecule according to claim 1 and an additional polypeptide.
 5. Thenucleic acid molecule of claim 4 wherein the additional polypeptide is aheterologous signal peptide.
 6. The nucleic acid molecule of claim 4wherein the additional polypeptide has adjuvant activity.
 7. The nucleicacid molecule according to claim 1, operatively linked to one or moreexpression control sequences.
 8. A vaccine comprising at least one firstnucleic acid according to claim 1, and a vaccine vector wherein eachfirst nucleic acid is expressed as a polypeptide, the vaccine optionallycomprising a second nucleic acid encoding an additional polypeptidewhich enhances the immune response to the polypeptide expressed by saidfirst nucleic acid.
 9. The vaccine of claim 8 wherein the second nucleicacid encodes an additional Chlamydia polypeptide.
 10. A pharmaceuticalcomposition comprising a nucleic acid according to claim 1 and apharmaceutically acceptable carrier.
 11. A pharmaceutical compositioncomprising a vaccine according to claim 8 and a pharmaceuticallyacceptable carrier.
 12. A unicellular host transformed with the nucleicacid molecule of claim
 7. 13. A nucleic acid probe of 5 to 100nucleotides which hybridizes under stringent conditions to the nucleicacid molecule of SEQ ID No: 1, or to a homolog or complementary oranti-sense sequence of said nucleic acid molecule.
 14. A primer of 10 to40 nucleotides which hybridizes under stringent conditions to thenucleic acid molecules of SEQ ID No: 1, or to a homolog or complementaryor anti-sense sequence of said nucleic acid molecule.
 15. A polypeptidecomprising an amino acid sequence selected from any one of: (a) SEQ IDNo: 2; (b) an immunogenic fragment comprising at least 12 consecutiveamino acids from a polypeptide of (a); and (c) a polypeptide of (a) or(b) which has been modified to improve its immunogenicity, wherein saidmodified polypeptide is at least 75% identical in amino acid sequence tothe corresponding polypeptide of (a) or (b).
 16. A fusion polypeptidecomprising the polypeptide of claim 15 and an additional polypeptide.17. The fusion polypeptide of claim 16 wherein the additionalpolypeptide is a heterologous signal peptide.
 18. The fusion protein ofclaim 16 wherein the additional polypeptide has adjuvant activity.
 19. Amethod for producing a polypeptide of claim 15, comprising the step ofculturing a unicellular host according to claim
 12. 20. An antibodyagainst the polypeptide of claim
 15. 21. A vaccine comprising at leastone first polypeptide according to claim 15 and a pharmaceuticallyacceptable carrier, optionally comprising a second polypeptide whichenhances the immune response to the first polypeptide.
 22. The vaccineof claim 21 wherein the second polypeptide comprises an additionalChlamydia polypeptide.
 23. A pharmaceutical composition comprising apolypeptide according to claim 15 and a pharmaceutically acceptablecarrier.
 24. A pharmaceutical composition comprising a vaccine accordingto claim 21 and a pharmaceutically acceptable carrier.
 25. Apharmaceutical composition comprising an antibody according to claim 20and a pharmaceutically acceptable carrier.
 26. A method for preventingor treating Chlamydia infection using the nucleic acid of claim
 1. 27. Amethod for preventing or treating Chlamydia infection using the vaccineof claim
 8. 28. A method for preventing or treating Chlamydia infectionusing the pharmaceutical composition of claim
 10. 29. A method forpreventing or treating Chlamydia infection using the polypeptide ofclaim
 15. 30. A method for preventing or treating Chlamydia infectionusing the antibody of claim
 20. 31. A method of detecting Chlamydiainfection comprising the step of assaying a body fluid of a mammal to betested with the nucleic acid of claim
 1. 32. A method of detectingChlamydia infection comprising the step of assaying a body fluid of amammal to be tested with the polypeptide of claim
 15. 33. A method ofdetecting Chlamydia infection comprising the step of assaying a bodyfluid of a mammal to be tested with the antibody of claim
 20. 34. Amethod for identifying the polypeptide of claim 15 which induces animmune response effective to prevent or lessen the severity of Chlamydiainfection in a mammal previously immunized with polypeptide, comprisingthe steps of: (a) immunizing a mouse with the polypeptide; and (b)inoculating the immunized mouse with Chlamydia; wherein the polypeptidewhich prevents or lessens the severity of Chlamydia infection in theimmunized mouse compared to a non-immunized control mouse is identified.35. Expression plasmid pCABk831.
 36. A nucleic acid molecule of SEQ IDNO. 3 or
 4. 37. An omp P6 precursor from Chlamydia.